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The Latest Research Items

Polypeptide Diblock Copolymers

The self-assembly of synthetic diblock copolymers has been extensively studied experimentally and theoretically. In contrast, self-assembly of polypeptide diblock copolymers has so far been mostly studied experimentally. Researchers in the Rubinstein Group, in collaboration with scientists from Duke University and the Russian Academy of Sciences, published in Macromolecules, have discovered that the theory developed for synthetic diblock copolymer does not fully explain the self-assembly of elastin-like polypeptide diblock copolymers, leading them to generalize the theory to make it applicable for these polypeptides.

The team, brought together through The Research Triangle MRSEC, demonstrated that elastin-like polypeptide diblocks self-assemble into weak micelles with dense cores and almost unstretched coronas, a state not previously observed for synthetic diblock copolymers. Weak micelles form if the surface tension at the core–corona interface is low compared to that expected of a micelle with a dense core. The predictions of the theory of weak micelles for the critical micelle temperature, hydrodynamic radius, and aggregation number of elastin-like polypeptide diblocks are in reasonable agreement with the experimentally measured values. The unique and unprecedented control of amphiphilicity in these recombinant peptide polymers reveals a new micellar state that has not been previously observed in synthetic diblock copolymer systems.

Photoinitiated Polymerization

Advances in controlled polymerization have revolutionized the synthesis of polymers of complex architecture for a variety of materials applications. Very recently, systems developed in the context of photoredox catalysis have been applied to controlled polymerization with encouraging results. In a collaboration between the You and Nicewicz research groups, published in JACS, a method for the visible light photoinitiated living cationic polymerization of 4-methoxystyrene using easy to synthesize, bench stable triarylpyrylium salts was developed. Careful study led to the discovery that inclusion of low concentrations of methanol imparted control over the polymerization, and allowed for the tuning of poly(4-methoxystyrene) molecular weight based on methanol concentration. Kinetic analysis, deuterium labeling studies, and monomer addition experiments revealed a living polymerization system wherein methanol imparts control in a manner analogous to dithiocarbamates in reversible addition-fragmentation chain transfer (RAFT) polymerization. Current work is underway to expand this method to a variety of monomers and towards the synthesis of block copolymers and other polymers of complex architecture.

Ribosome Structure Resolved

It has been known for decades that the ribosome, the cellular complex that synthesizes proteins, interconverts between "active" and "inactive" conformations. However, the physiological relevance of this widely observed switch remained unclear and unknown.

Jennifer McGinnis, in the Weeks Lab, led a study, published in PNAS, in which newly developed in-cell SHAPE technologies were used to probe the structure of the ribosome in healthy living cells. In cells, one class of ribosome subunits exists predominantly in the classic "inactive" conformation and disrupting the ability to interconvert between active and inactive conformations compromises protein synthesis. In-cell RNA structure probing thus resolved this 40 year old challenge to reveal that the inactive state regulates ribosome function as a conformational switch.

Hydrogen Exchange

A truly disordered protein lacks a stable fold and its backbone amide protons exchange with solvent at rates predicted from studies of unstructured peptides. In a paper published in Protein Science, Austin Smith, a graduate student in the Pielak Group have measured the exchange rates of two model disordered proteins, FlgM and α-synuclein, in buffer and in Escherichia coli using the NMR experiment, SOLEXSY.

The rates are similar in buffer and cells and are close to the rates predicted from data on small, unstructured peptides. This result indicates that true disorder can persist inside the crowded cellular interior and that weak interactions between proteins and macromolecules in cells do not necessarily affect intrinsic rates of exchange. Parenthetically, the research Austin did for this paper earned him the Best Poster Award at the Protein Society's annual conference last year.

Degradation Mechanisms

The decomposition of molecular catalysts can limit the lifetime of their activity and hinder mechanistic study. Given the tremendous effort applied to developing new catalysts for areas of societal importance, including sunlight-to-fuel schemes, identifying when catalysts are decomposing, understanding the mechanisms by which catalysts decompose, and recognizing what structural factors predispose a catalyst towards degradation is of importance. To date, most work has focused on identifying only if a molecular complex degrades into a catalytically competent heterogeneous species, a non-trivial task.

Published as an "Edge" article in Chemical Science, researchers in the Dempsey Group show how a Ni(II) bisphosphine dithiolate compound degrades into an electrode-adsorbed film that can evolve hydrogen under reducing and protic conditions. Their electrochemical study suggests that the degradation mechanism involves an initial concerted proton–electron transfer. Also, the paper brings up the potential susceptibility of Ni–S bonds in molecular hydrogen evolution catalysts to degradation via C–S bond cleavage.

Thiomarinol Biosynthesis

Thiomarinol is a naturally occurring double-headed antibiotic that is highly potent against methicillin-resistant Staphylococcus aureus. Its structure comprises two antimicrobial subcomponents, pseudomonic acid analogue and holothin, linked by an amide bond. TmlU was thought to be the sole enzyme responsible for this amide-bond formation. In contrast to this idea, researchers in the Li Group, published in Angewandte Chemie, we show that TmlU acts as a CoA ligase that activates pseudomonic acid as a thioester that is processed by the acetyltransferase HolE to catalyze the amidation.

 

Research Image

TmlU prefers complex acyl acids as substrates, whereas HolE is relatively promiscuous, accepting a range of acyl-CoA and amine substrates. The Li group's results provide detailed biochemical information on thiomarinol biosynthesis, and evolutionary insight regarding how the pseudomonic acid and holothin pathways converge to generate this potent hybrid antibiotic. Their work also demonstrates the potential of TmlU/HolE enzymes as engineering tools to generate new "hybrid" molecules.

 

Copper in Water Oxidation

Copper metal is in theory a viable oxidative electrocatalyst based on surface oxidation to CuIII and/or CuIV, but its use in water oxidation has been impeded by anodic corrosion. Researchers from the Meyer Group, published in Angewandte Chemie, present the in situ formation of an efficient interfacial oxygen-evolving Cu catalyst from CuII in concentrated carbonate solutions.

Research Image

The catalyst necessitates use of dissolved CuII and accesses the higher oxidation states prior to decompostion to form an active surface film, which is limited by solution conditions. This observation and restriction led to the exploration of ways to use surface-protected Cu metal as a robust electrocatalyst for water oxidation. Formation of a compact film of CuO on Cu surface prevents anodic corrosion and results in sustained catalytic water oxidation. The Cu/CuO surface stabilization was also applied to Cu nanowire films, which are transparent and flexible electrocatalysts for water oxidation and are an attractive alternative to ITO-supported catalysts for photoelectrochemical applications.

 

Sphingosine Kinase Activity

Sphingosine-1-phosphate, S1P, a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase ,SK, plays a crucial role in natural killer, NK, cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention.

However, peripheral blood NK cells consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In a collaborative work, published as a cover article in Integrative Biology, researchers in the Allbritton Group, measured the phosphorylation of sphingosine–fluorescein, SF, and subsequent metabolism of S1P fluorescein, S1PF, to form hexadecanoic acid fluorescein, HAF, in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. Substantial heterogeneity in S1P production and metabolism across cells within and between subjects was readily apparent. NK-cell subpopulations may exist with respect to SK activity and individual humans may possess distinct phenotypes. A deeper understanding of lipid signaling at the single-cell level will be critical to understand NK cell biology and disease.

Aerosol Analysis

Low-temperature plasma ionization, a technique that causes minimal fragmentation during ionization, has been investigated by the Glish Group as an ionization technique for mass spectrometric detection of the compounds in ambient organic aerosols in real time.

Research Images

The experiments presented in a paper published in Analytical Chemistry demonstrate that ions are generated from compounds in the aerosol particles. The utility of this technique for detection of both positive and negative ions from the pyrolysate of multiple natural polymers is presented. Ultimately, low-temperature plasma ionization is shown to be a promising ionization technique for detection of compounds in organic aerosols by mass spectrometry.

 

Hopping Diffusion of Nanoparticles

In a collaborative work, published in Macromolecules, researchers in the Rubinstein Group propose a hopping mechanism for diffusion of large nonsticky nanoparticles subjected to topological constraints in both unentangled and entangled polymer solids, networks and gels, and entangled polymer liquids, melts and solutions. Probe particles with size larger than the mesh size ax of unentangled polymer networks or tube diameter ae of entangled polymer liquids are trapped by the network or entanglement cells. At long time scales, however, these particles can diffuse by overcoming free energy barrier between neighboring confinement cells.

Research Images

The terminal particle diffusion coefficient dominated by this hopping diffusion is appreciable for particles with size moderately larger than the network mesh size ax or tube diameter ae. Much larger particles in polymer solids will be permanently trapped by local network cells, whereas they can still move in polymer liquids by waiting for entanglement cells to rearrange on the relaxation time scales of these liquids. Hopping diffusion in entangled polymer liquids and networks has a weaker dependence on particle size than that in unentangled networks as entanglements can slide along chains under polymer deformation. The proposed novel hopping model enables understanding the motion of large nanoparticles in polymeric nanocomposites and the transport of nano drug carriers in complex biological gels such as mucus.

 

In-Cell Ribosome Structure Resolved

It has been known for decades that the ribosome, the cellular complex that synthesizes proteins, interconverts between "active" and "inactive" conformations. However, the physiological relevance of this widely observed switch remained unclear and unknown.

Research Images

Jennifer McGinnis, in the Weeks Lab, led a study, published in PNAS, in which newly developed in-cell SHAPE technologies were used to probe the structure of the ribosome in healthy living cells. In cells, one class of ribosome subunits exists predominantly in the classic "inactive" conformation and disrupting the ability to interconvert between active and inactive conformations compromises protein synthesis. In-cell RNA structure probing thus resolved this 40 year old challenge to reveal that the inactive state regulates ribosome function as a conformational switch.

 

Fine Chemicals

Traditional strategies to access fine chemicals require the use of non-renewable fossil fuel sources. Biorenewable materials, such as carbohydrates would offer an alternative inexpensive and abundant feedstock source that could reduce the current reliance on fossil fuels for these chemicals. Although they are potentially valuable chemical feedstocks, their high oxygen content renders them "overfunctionalized" for many synthetic applications. Researchers in the Gagné Group have recently published an article in Nature Chemistry, where it was also included as a News & Views article describing methods to selectively reduce some of the hydroxyl content while preserving enough stereocenters to provide useful chiral synthons to furnish fine chemicals. In this procedure, a commercially available boron species activates a trialkylsilane into a Lewis acidic R3Si+ equivalent and an H- reducing agent. Well-defined cyclic species are implicated in the efficient and controlled reduction of these substrates, allowing access to a variety of chemical building blocks that are new or otherwise difficult to produce.

Microchip HDX/MS

Hydrogen/deuterium exchange mass spectrometry (HDX MS) is a powerful tool for investigating the conformation and dynamics of intact proteins. The current paradigm of HDX MS relies on LC separations performed at low temperatures with fast gradient times in order to minimize hydrogen/deuterium back-exchange. Researchers in the Ramsey Group, published in Analytical Chemistry, developed a microchip capillary electrophoresis-electrospray ionization device for fast and high efficiency HDX MS analysis of proteins. The high speed CE-ESI of a bovine hemoglobin digestion was performed in 1 min compared to 7 min with a similar LC separation and with nearly triple the peak capacity.

Hydrogen Exchange of Disordered Proteins

A truly disordered protein lacks a stable fold and its backbone amide protons exchange with solvent at rates predicted from studies of unstructured peptides. In a paper published in Protein Science, Austin Smith, a graduate student in the Pielak Group have measured the exchange rates of two model disordered proteins, FlgM and α-synuclein, in buffer and in Escherichia coli using the NMR experiment, SOLEXSY.

Research Image

The rates are similar in buffer and cells and are close to the rates predicted from data on small, unstructured peptides. This result indicates that true disorder can persist inside the crowded cellular interior and that weak interactions between proteins and macromolecules in cells do not necessarily affect intrinsic rates of exchange. Parenthetically, the research Austin did for this paper earned him the Best Poster Award at the Protein Society's annual conference last year.

 

Degradation Mechanisms

The decomposition of molecular catalysts can limit the lifetime of their activity and hinder mechanistic study. Given the tremendous effort applied to developing new catalysts for areas of societal importance, including sunlight-to-fuel schemes, identifying when catalysts are decomposing, understanding the mechanisms by which catalysts decompose, and recognizing what structural factors predispose a catalyst towards degradation is of importance. To date, most work has focused on identifying only if a molecular complex degrades into a catalytically competent heterogeneous species, a non-trivial task.

 

Research Image

Published as an "Edge" article in Chemical Science, researchers in the Dempsey Group show how a Ni(II) bisphosphine dithiolate compound degrades into an electrode-adsorbed film that can evolve hydrogen under reducing and protic conditions. Their electrochemical study suggests that the degradation mechanism involves an initial concerted proton–electron transfer. Also, the paper brings up the potential susceptibility of Ni–S bonds in molecular hydrogen evolution catalysts to degradation via C–S bond cleavage.

 

Stereocontrolled Total Synthesis of Paspaline

Robert Sharpe, a fourth year graduate student in the Johnson Group, describes in an article published in JACS, the stereocontrolled total synthesis of the indole diterpenoid natural product paspaline.

Research Image

Key steps in the process include a highly diastereoselective enzymatic desymmetrization, substrate-directed epoxidation, Ireland-Claisen rearrangement, and diastereotopic group selective C–H acetoxylation to assemble the target with excellent stereofidelity. The route and results described in the paper outline complementary conceptual disconnections in the arena of steroid natural product synthesis.

 

Thiomarinol Biosynthesis

Thiomarinol is a naturally occurring double-headed antibiotic that is highly potent against methicillin-resistant Staphylococcus aureus. Its structure comprises two antimicrobial subcomponents, pseudomonic acid analogue and holothin, linked by an amide bond. TmlU was thought to be the sole enzyme responsible for this amide-bond formation.

In contrast to this idea, researchers in the Li Group, published in Angewandte Chemie, show in a collaborative work that TmlU acts as a CoA ligase that activates pseudomonic acid as a thioester that is processed by the acetyltransferase HolE to catalyze the amidation.

TmlU prefers complex acyl acids as substrates, whereas HolE is relatively promiscuous, accepting a range of acyl-CoA and amine substrates. The Li group's results provide detailed biochemical information on thiomarinol biosynthesis, and evolutionary insight regarding how the pseudomonic acid and holothin pathways converge to generate this potent hybrid antibiotic. Their work also demonstrates the potential of TmlU/HolE enzymes as engineering tools to generate new "hybrid" molecules.

Analysis of Sphingosine Kinase Activity

Sphingosine-1-phosphate, S1P, a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase ,SK, plays a crucial role in natural killer, NK, cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention.

Research Image

However, peripheral blood NK cells consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In a collaborative work, published as a cover article in Integrative Biology, researchers in the Allbritton Group, measured the phosphorylation of sphingosine–fluorescein, SF, and subsequent metabolism of S1P fluorescein, S1PF, to form hexadecanoic acid fluorescein, HAF, in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. Substantial heterogeneity in S1P production and metabolism across cells within and between subjects was readily apparent. NK-cell subpopulations may exist with respect to SK activity and individual humans may possess distinct phenotypes. A deeper understanding of lipid signaling at the single-cell level will be critical to understand NK cell biology and disease.

 

Copper in Water Oxidation

Copper metal is in theory a viable oxidative electrocatalyst based on surface oxidation to CuIII and/or CuIV, but its use in water oxidation has been impeded by anodic corrosion. Researchers from the Meyer Group, published in Angewandte Chemie, present the in situ formation of an efficient interfacial oxygen-evolving Cu catalyst from CuII in concentrated carbonate solutions.

The catalyst necessitates use of dissolved CuII and accesses the higher oxidation states prior to decompostion to form an active surface film, which is limited by solution conditions. This observation and restriction led to the exploration of ways to use surface-protected Cu metal as a robust electrocatalyst for water oxidation. Formation of a compact film of CuO on Cu surface prevents anodic corrosion and results in sustained catalytic water oxidation. The Cu/CuO surface stabilization was also applied to Cu nanowire films, which are transparent and flexible electrocatalysts for water oxidation and are an attractive alternative to ITO-supported catalysts for photoelectrochemical applications.

Aerosol Analysis

Low-temperature plasma ionization, a technique that causes minimal fragmentation during ionization, has been investigated by the Glish Group, as an ionization technique for mass spectrometric detection of the compounds in ambient organic aerosols in real time. The experiments presented in a paper published in Analytical Chemistry demonstrate that ions are generated from compounds in the aerosol particles. The utility of this technique for detection of both positive and negative ions from the pyrolysate of multiple natural polymers is presented. Ultimately, low-temperature plasma ionization is shown to be a promising ionization technique for detection of compounds in organic aerosols by mass spectrometry.

Multicolor Monitoring

The proteasome, a validated anticancer target, participates in an array of biochemical activities, which range from the proteolysis of defective proteins to antigen presentation. Researchers in the Lawrence Group, report in ACS Chemical Biology, on the preparation of biochemically and photophysically distinct green, red, and far-red real-time sensors designed to simultaneously monitor the proteasome's chymotrypsin-, trypsin-, and caspase-like activities, respectively.

Research Images

These sensors were employed to assess the effect of simultaneous multiple active site catalysis on the kinetic properties of the individual subunits. Furthermore, the team found that the catalytic signature of the proteasome varies depending on the source, cell type, and disease state. Trypsin-like activity is more pronounced in yeast than in mammals, whereas chymotrypsin-like activity is the only activity detectable in B-cells, unlike other mammalian cells. Furthermore, chymotrypsin-like activity is more prominent in transformed B cells relative to their counterparts from healthy donors.